First generation nucleic acid biochips are very expensive to produce, requiring large capital investments, process engineering and equipment. Furthermore, the method of forming oligonucleotides in a single layer on a substrate results in a low sensitivity biochip often requiring an expensive laser confocal fluorescence microscope for adequate detection of DNA specifically hybridized to the chip. U.S. Pat. No. 5,744,305 is an example of such a biochip.
In view of the low sensitivity of these first generation biochips, second generation biochips have been developed, such as those described in U.S. Pat. Nos. 5,736,257 and 5,847,019.
The biochips of the '257 and '019 patents employ a polyacrylamide network on top of a molecular layer of vinyl groups, thereby giving a third dimension to the gel cells. Still, as will be readily appreciated by those of skill in the art, production of biochips in accordance with the disclosures of the '257 and '019 patents is not only expensive but also time-consuming.
U.S. Pat. No. 5,552,270, issued to Khrapko, et al., describes a method of sequencing DNA which utilizes a second generation biochip comprising a solid support and a matrix that includes an array of oligonucleotides of desired lengths is attached to the support by means of a gel layer having a thickness of 30 μm or less.
Another polyacrylamide-based biochip described U.S. Pat. No. 5,770,721 is based upon the polymerization of acrylamide monomers by a free radical initiation or ultraviolet radiation process; however, this polyacrylamide-based gel biochip is constructed in a multi-step process that is lengthy and labor-intensive. Production of such a biochip requires cumbersome multi-step processing including polymerization and binding to the surface of the glass substrate; mechanical or laser cutting to form micro-squares of gel matrix on the substrate; activation step using hydrazines; and finally reaction with the oligonucleotides. Due to the polymerization process of inherent polyacrylamide gels, these steps must be performed independently. Thus, the total time required to produce a single biochip by such methods is at least about 24 to 48 hours. Furthermore, after each step, thorough washings and/or other special cares must be taken before the next step is begun. For example, the oligonucleotide derivatization step requires a long incubation period, such as twenty-four to forty-eight hours. Moreover, the potential reaction of the oligonucleotides with the hydrazine groups would form unstable morpholine derivatives, resulting in a very short shelf half-life for the biochip of approximately thirty-six hours at room temperature. Thus, there is a significant need for a simple, cost effective, rapid method for constructing a reliable multi-functional nucleic acid biochip having high sensitivity and a reasonably long shelf-life that may be used in gene discovery, gene characterization, functional gene analysis and related studies.
From the standpoint of studying protein-ligand, protein-protein, protein-DNA interaction, there are presently a number of known methods, all of which possess significant limitations in that they are either cumbersome, expensive, require large amounts of proteins or are not suitable for the rapid high throughput analysis of protein interactions.
An early method used to study protein interactions is the protein-affinity column. With this method, the capture protein is covalently immobilized to agarose beads and used to affinity-purify a target protein from a heterogeneous mixture containing many contaminating proteins through the use of affinity chromatography. This method requires relatively large amounts of capture proteins for suitable immobilization to agarose beads, and it is not suitable for the rapid high throughput screening of protein interactions.
A further method used to study protein interactions is the yeast 2-hybrid system. With this method, a target protein library is constructed in yeast. This system is designed to express these proteins of interest, with each being linked to a transcription activating region. DNA encoding for a bait protein (or a protein being examined for possible other interacting proteins) is fused to a DNA binding domain and is also expressed in the same library. A reporter gene carrying the corresponding DNA sequence is also included which codes for a detection system, such as a fluorescent protein or a protein with easily detectable biological activities. Upon binding of the target protein of interest to the bait protein, the consequent interaction of the two achieves activation of the reporter gene and results in signal generation. Even though this method may relatively frequently be used, it is slow and cumbersome, requires significant molecular biology expertise, and does not lend itself to the rapid, high throughput screening of protein-protein interaction in an economical way.
Another early method commonly employed to study protein interactions is immunoprecipitation of both the capture and the target protein, followed by analysis of the resulting complex using polyacrylamide gel electrophoresis. With this method, the capture protein is first incubated with a heterogeneous mixture of proteins allowing it to bind to its target. The resulting complex is then immunoprecipitated using antibodies raised against one protein of the pair, and the complex is separated for analysis by gel electrophoresis and followed by a detection step, e.g. staining by dye. This method is slow and cumbersome, requires significant biochemistry expertise, and likewise does not lend itself to the rapid high throughput analysis of protein interactions.
Another method used to study protein interactions is phage display. With this method, a library of proteins is expressed on the flagella of certain filamentous phage proteins expressed on the surface of a host bacterium, e.g. E. coli so as to provide an affinity support for such “displayed” proteins. The phage library is then exposed to a number of potential target proteins. The binding of the displayed protein to the target protein allows target identification. This method has a number of limitations, e.g. large molecular weight proteins are difficult to display, and only very few of a phage's filamentous proteins are appropriate for such use. In addition, conformational constrictions of the displayed protein have been known to decrease its affinity and consequently affect its ability to bind to its natural ligand.
The fabrication of a high-throughput-capable microarray or biochip suitable for binding such entities, e.g. proteins, will generally require the use of a method to attach proteins to the surface in a manner so that they may thereafter be used for detection by readily interacting with other materials or molecules of interest, e.g. proteins, commonly referred to as targets. For example, proteins may be bound directly to a surface treated with divalent or trivalent metal ions, such as Cu2+ or Fe3+, to which proteins will naturally bind with varying degrees of affinity. If targets then bind to the probes, they can be detected and identified by SELDI™ (surface-enhanced laser desorption/ionization) in combination with a mass spectrometer, as described in U.S. Pat. No. 5,719,060. In an alternative method described by G. MacBeath and S. Schreiber. (Science 289:1760, 2000), chemical binding is also used to attach proteins to a substrate surface, while target ligands are labeled with fluorescence tags; thus, any interaction between probes and labeled targets can be detected using a fluorescence-based slide scanner.
Because the above methods of protein immobilization to provide binding entities generally employ direct chemical conjugation of proteins onto the surface of a substrate, these methods embody a major limitation in resultant loss of protein function, due either to inappropriate chemical conjugation at active sites or to loss of original conformation. When such occurs, only a low amount of immobilized protein remains active and results in detection difficulties and low assay sensitivity. In addition, the complexity and lack of precision of these methods generally render them unsuitable for use in fabrication of high-density microarrays for high throughput use.
U.S. Pat. No. 6,087,102 describes a method which utilizes a polyacrylamide gel to create individual cells, composed of the electrophoresed protein spots, which can be subsequently crosslinked in situ into the gel to form a biochip. Limitations of the method include difficulties in preparing precise, small cells on the biochip and in potential destructive effects on the capture protein during crosslinking. U.S. Pat. No. 5,847,019 describes another approach which utilizes photopolymerizable polymers to form a patterned network layer to fabricate a biochip, using light-reactive free-radical chemistry. This photoactivation approach used for the immobilization of proteins to a biochip appears limited to certain photoactivation chemistries involving acrylamide polymers, and moreover, the use of free radical photochemistry may cause potential free radical damage to the capture proteins being used in biochips fabricated in such fashion.
The use of isocyanate-capped liquid polyurethane prepolymers to directly react with proteins to immobilize proteins within polyurethane foams is described in U.S. Pat. Nos. 4,098,645 and 3,672,955 which teach the use of isocyanate-functional hydrogel systems to bind proteins directly through their amino and hydroxyl sites to thereby form enzyme reactors and antibody/antigen based affinity columns. While the described methodology may be suitable for such purposes, these processes do not form optically clear hydrogels of controlled geometry which would be suitable for biochip use. Additionally, using such methods without inhibiting potential conjugation to protein side chains may very likely cause undesirable crosslinking of the protein to the polymer, and extensive crosslinking may diminish or destroy the native conformation of the protein and thus reduce the bioactivity of the protein which would render such process unsuitable for high precision binding assays for which biochips are used.
Despite these technical hurdles, the importance of understanding protein-protein and other comparable biomolecular interactions has made the achievement of a practical, flexible format biochip, suitable for incorporation of a number of different nonhybridization binding entities, a desired tool for a variety of research and commercial applications in biological science. In short, there exists a need for an efficient anchoring or support system which will support such entities in a manner such that they retain maximal binding activity, so as to allow for the construction of microarrays that would parallel nucleic acid arrays.
Thus, it is desirable to provide improved methods making nucleic acid biochips, as well as methods for enabling binding entities, such as proteins, to be immobilized or encapsulated in a manner which allows them to retain their native conformation and function so that they would be free to sequester targets.